Splicing is essential and prevalent in humans. Human genes in are interrupted on average by eight introns. Moreover, nearly 95% of human genes are alternatively spliced at least 15% of the time, providing a versatile mechanism to regulate gene expression quantitatively and qualitatively. Not surprisingly then, at least 15% of human diseases derive from defects in pre-mRNA splicing. Yet, our mechanistic understanding of the spliceosome remains primitive. In the spliceosome, a ribonucleoprotein machine composed of five small nuclear RNAs (snRNAs) and 80 conserved proteins, the snRNAs play key roles in both substrate recognition and likely catalysis. Throughout the splicing cycle the snRNA components undergo dramatic rearrangements thought to require eight conserved members of the DExD/H box family of ATPases. Our long-term goal is to understanding the function and mechanism of the DExD/H box ATPases and the snRNA rearrangements they catalyze. In this proposal, we aim to capitalize on our recent advances. First, by establishing unique assays for the critical but poorly understood fidelity activities in splicing, we have found evidence in vitro that each chemical step in splicing is proofread by a specific DExD/H box ATPase to reject suboptimal substrates and further that a distinct DExD/H box ATPase functions at both chemical steps to discard rejected substrates. However, it remains unclear how these DExD/H box ATPases permit discrimination between optimal and suboptimal substrates and whether kinetic proofreading plays a role. Second, we have also implicated a dual role for the DExD/H box ATPase Brr2 and regulation of Brr2 by a GTPase and ubiquitin, but the mechanism and utility of these switches in the splicing cycle remains to be investigated. Third, the DExD/H box ATPase Prp2 is the last ATPase required in spliceosome activation, but the function of this ATPase in promoting the catalytic conformation of the spliceosome remains enigmatic. Through our studies of the role of the DExD/H box ATPase Prp16 in rearranging the catalytic conformation of the spliceosome, we have gained insight into the snRNA configuration of the catalytic conformation and potential consequences of Prp2-dependent RNA rearrangements. Thus, the aims of this proposal are i) to define the ATP dependent mechanism for establishing fidelity during exon ligation, ii) to investigate a role for ubiquitin in regulating Brr2-dependent spliceosome activation and disassembly and iii) to investigate the role of Prp2 in spliceosome activation. We will pursue these aims using a combined approach of genetics and biochemistry in budding yeast, while exploiting emerging single molecule and next generation sequencing technologies. Through these studies, we expect to advance our understanding of the role of DExD/H box ATPases in fidelity, the mechanisms for regulating these ATPases, and the function of these ATPases in rearranging noncoding RNA. Further, given our recent implication of hBrr2-defective RNA unwinding in the etiology of autosomal dominant retinitis pigmentosa, this work will also have important implications for this common form of blindness.